Hyaluronic acid is a naturally occurring high molecular weight polysaccharide typically recovered as its sodium salt having an empirical formula of (C.sub.14 H.sub.20 N Na O.sub.11).sub.n where n&gt;1000. The general structure of hyaluronic acid is illustrated in Merck Index, Ninth Ed. (3rd printing, 1978), at page 624. It is well known that hyaluronic acid and its salts, hereafter collectively referred to as HA, can be obtained from at least three sources: human umbilical cords, rooster combs and certain bacterial cultures such as group A and C hemolytic streptococci. However, certain disadvantages are associated with the former two sources (e.g. relatively low yields, contamination with chondroitin sulfate, and labor intensive processing and purification steps).
Since HA is found in aqueous and vitreous humor of eyes and the synovial fluid of mammalian joints, there has been considerable interest in obtaining purified HA for use as a fluid replacement to correct pathological conditions in the eye and in joints. The preparation of HA from rooster combs and human umbilical cords and its use in eye and joint applications is described in U.S. Pat. No. 4,141,973 to E. A. Balazs. That patent also provides a detailed review of the technical literature describing the isolation, characterization and uses of HA.
U.S. Pat. No. 4,303,676, also to E. A. Balazs, describes cosmetic formulations containing sodium hyaluronate fractions in various molecular weight ranges made from rooster combs. U.S. Pat. No. 4,328,803 to L. G. Pape discloses the use of an ultrapure hyaluronic acid salt in eye surgery. The HA product used was a sodium hyaluronate salt available under the registered trademark HYLARTILB.RTM. from Pharmacia, Inc. and obtained in commercial quantities from rooster combs.
Because the medical applications of HA require that the HA be injected into a mammalian body (e.g. as a fluid replacement), it is very important that the injected products be as pure as possible to avoid reactivity problems. This importance of purity is described in U.S. Pat. No. 4,141,973 which describes an ultrapure HA product prepared from rooster combs or, alternatively, from human umbilical cords. In addition to purity, it appears that control of molecular weight of an HA product is very important (e.g. the U.S. Pat. No. 4,141,973 suggests an average molecular weight of at least 750,000 daltons and U.S. Pat. No. 4,303,676 suggests having two distinct fractions of controlled molecular weight, one low and one high). Although there is a description of a high molecular weight (1,200,000 daltons) HA preparation of very high purity (i.e. less than 0.05% protein) in a paper by Swann, Arch. Opthal. 88, pp. 544-8 (1972), we are unaware of any description of an HA product having the following advantages: (1) derivable from a microbiological source at relatively low costs, in high yields, and with low reactivity upon injection; (2) having a desirably high and closely controlled average molecular weight; and (3) being substantially free of protein and nucleic acid impurities.
The microbiological production of hyaluronic acid is well known in the literature. A rather extensive discussion is found in "The Biosynthesis of Hyaluronate by Group A Streptococci", a 1955 doctoral thesis on file at The University of Minnesota. Japanese Published Patent Application 83-56692 teaches greatly enhancing the production of hyaluronic acid from Streptococcus zooepidemicus and Streptococcus equi cultures by adding high levels of glucose to a protein (yeast extract) containing culture and continuously aerating while shaking. Although a 1976 paper of Kjem in Acta. Pathol.Microbial Scand. is acknowledged as teaching microbiological production of hyaluronic acid no explicit mention of a chemical defined medium (CDM) or a protein free medium is made. There is no indication that its technology could be applied to such a specialized medium. U.S. Pat. No. 2,975,104 to Warren teaches a technique for increasing the acceptable incubation time of streptococci in producing hyaluronic acid by the use of a particular medium which contains a hyaluronidase inhibitor. The growth of Group A streptococcal strains in a chemically defined medium (CDM) with the production of a hyaluronic acid capsule is disclosed in "Growth Characteristics of Group A Streptococci in a New Chemically Defined Medium" by Van de Rijn and Kessler at pages 444 to 448 of Volume 27, Number 2 (February 1980) of Infection and Immunity. The recovery of hyaluronic acid from a somewhat different CDM by heat killing the culture, filtering the medium, precipitating with ethanol, centrifuging the precipitate, suspending the precipitate in an aqueous sodium chloride solution, reprecipitating with cetyl pyridinium chloride, centrifuging the reprecipitate, dissolving this reprecipitate in an aqueous sodium chloride solution and finally precipitating once again with ethanol is described in "Isolation of Hyaluronic Acid from Cultures of Streptococci in a Chemically Defined Medium" by Kjems and Lebech at pages 162 to 164 of Section B, Volume 84 (1976) of Acta Path. Microbial. Scand. This procedure has some significant disadvantages. The heat killing of the microorganisms will result in the hyaluronic acid's becoming unnecessarily contaminated with proteins, nucleic acids and other internal cell components which are difficult to separate from the hyaluronic acid and can provoke immune reactions in mammals. Thus, some of the benefit of utilizing a chemically defined medium to avoid protein contamination and achieve nonantigenicity is unnecessarily lost as compared to the contamination suffered merely from the natural death of cells in a growing culture. Furthermore, this CDM itself has limited utility because it will not support the growth of most streptococcal strains. The recovery of hyaluronic acid of a mean molecular weight of about 55,000 daltons from an anaerobically grown culture of Streptococcus pyogenes inactivated with trichloroacetic acid by filtering using a 0.22 micrometer pore size, disfiltering with a filter having a nominal retention of 30,000 daltons molecular weight until the conductivity of the filtrate is 0.5 mesa-ohms (believed to mean inverse ohms per centimeter times 10), precipitating with ethanol, resuspending in an aqueous sodium chloride solution, precipitating with CETAB (believed to be hexadecyl trimethyl ammonium bromide), coarse filtering, resuspending in an aqueous sodium chloride solution and disfiltering with a filter having a nominal molecular weight retention of 30,000 daltons until the conductivity is "0.5 mesa-ohms" is described in U.S. Pat. No. 4,517,295 to Bracke and Thacker. This procedure also involves the troublesome killing of the culture cells. In this case, the trichloroacetic acid not only kills these cells with the resultant contamination problem, but it also is relied on for an effective separation of the cells from the broth. Furthermore, this broth is only semi-defined and contains casein derived proteins which may also contaminate the obtained hyaluronic acid.
Non-antigenic hyaluronic acid is also discussed in the literature. A process of obtaining hyaluronic acid free from proteins, antigens and pyrogens by treating an aqueous alkaline suspension to denature the protein, subjecting the suspension under appropriate conditions to proteolytic ferments, removing amino acids and mineral salts with ion exchangers, and aciditying to pH 3 to 4 to precipitate the remaining impurities with some of the hyaluronic acid is described in U.S. Patent No. 3,396,081 to Billek. A reportedly ultrapure hyaluronic acid suitable for injection into the human eye or animal joints and obtained by an involved extraction procedure including a five day chloroform extraction under mixing is described in U.S. Pat. No. 4,141,973 to Balazs.
A useful procedure for the isolation of hyaluronic acid capsules from streptococci cultures by incubating the bacteria, which had already been isolated by centrifugation and washed with saline, with sodium dodecyl sulfate in a saline suspension until the capsule was released, centrifuging to recover the supernatant, filtering the supernatant through a 0.22 micrometer pore filter, precipitating the hyaluronic acid with hexadecyltrimethyl ammonium bromide, recovering the precipitate by centrifugation, redissolving the precipitate in 2M calcium chloride and clarifying the solution by centrifugation, precipitating with ethanol, redissolving with water followed by the addition of sodium chloride and clarifying the solution by centrifugation, and repeating the ethanol precipitation five times is described in "Streptococcal Hyaluronic Acid: Proposed Mechanism of Degradation and Loss of Synthesis During Stationary Phase" by Van de Rnn at pages 1059 to 1065 of volume 156, number 3 (December, 1983) of the Journal of Bacteriology. The use of sodium dodecyl sulfate to separate another virulence factor, M-protein, from Streptococcus equi is discussed in U.S. Pat. No. 4,582,798 to Brown, Bryant and Lewis. These latter two developments became publicly available after many of the developments with which the present disclosure is concerned.
There is a need for an efficient and inexpensive procedure to obtain high molecular weight hyaluronic acid suitable for injection into mammals. A microbiological fermentation which gives high yields of a relatively uncontaminated high molecular weight hyaluronic acid and a recovery procedure which does not create unnecessary purification problems and does not adversely affect the molecular weight of the hyaluronic acid are both desirable and would meet this need.